The long term goals of this proposal are to develop a detailed understanding of how retroviral DNA gains access to its integration targets in the host chromosome, and how the integration reaction is completed in the cell. The avian sarcoma/leukosis virus (ASV) is the primary model for these studies. However, use of an ASV derivative that can integrate its DNA into mouse and human, allows - access to the broad base of genetic and biochemical information available for mammalian systems, and - direct comparisons with murine (MLV) and human (HIV-1) retroviruses. Two complementary Specific Aims will be pursued. In Specific Aim 1 host proteins that facilitate nuclear entry of the ASV pre-integration complex and its association with host chromatin will be identified and characterized: (a) Systems to monitor the effects of the cell cycle on retroviral replication and active nuclear import of viral DNA will be developed and used - to compare the critical features of ASV with MLV and HIV- 1 and, - to investigate the role of the ASV integrase (IN) nuclear localization signal (NLS) in mediating the nuclear entry of viral DNA. (b) Cellular proteins that interact with the ASV IN NLS to facilitate nuclear entry will be identified and characterized and, (c) The hypothesis that an ASV IN-interacting cellular protein (Daxx) has a role in integration will be tested. Specific Aim 2 will investigate the role of host cell functions in retroviral DNA integration and is based on recent genetic evidence from this laboratory implicating the cellular, non-homologous end-joining (NHEJ) DNA repair pathway in retroviral DNA integration (Daniel eta!., Science 284: 644, 1999): (a) The hypotheses that - joining of the 3'-end of viral DNA to host DNA is required to elicit an NHEJ response and - components of NHEJ are required for subsequent joining of the 5'-ends of viral to host DNA, will be investigated; (b) Physical and functional interactions between retroviral and NHEJ components will be analyzed and, (c) The question of how early events leading to retroviral DNA integration may affect other proteins will be addressed using proteomics. A wide range of state-of-the-art genetic, biochemical, and cell biological methodologies will be employed. Retroviral DNA integration is an essential step in the replication cycle of retroviruses; it also contributes significantly to their pathogenicity. With a focus on virus-host cell interactions, these studies will reveal molecular mechanisms relevant to both retroviral and cellular biology, including nuclear import and cellular functions that are critical for genome stability. The studies may also suggest new strategies to prevent or treat retroviral disease by targeting cellular as well as viral functions.